It is frequently the case that it is desired to analyze molecular species, and charged protein species in particular, that are present in very low concentration either because the sample itself is very small or dilute or because the species of interest is present as a consequence of prior chemical processing and is thus at very low concentration. Moreover, these molecular species can be charged and being in the presence of uncharged molecules present further difficulty in analysis or separation.
Prior art provides a plurality of methods for concentrating molecular species from solution. However, there are numerous problems associated with these prior methods, such as the need for specialized column packing, the need for specialized solvents or buffer solutions, the need to change solvents or buffer solutions in order to elute concentrated molecular species, the need to change flow direction or flow conditions between the steps of retaining and eluting species, and the inability to either separate charged and uncharged molecular species or effect an efficient separation.
Zare et al. (Anal. Chem., 73, 3921–3926, 5539–5543, 5557–5563, November 2001) describe porous sol-gel monoliths that overcome some of the problems inherent in prior art column packing materials. These monoliths, useful for capillary electrochromatography, wherein neutral species are to be separated, can be prepared by a one-step process but their pore structure is uncontrolled.
Palm et al., Anal. Chem., 69, 4499–4507, 1997, have described a one-step process for in situ preparation of macroporous polyacrylamide gel matrices for capillary electrochromatography that can be purged by the use of electroosmotic flow (EOF). While the solvent can be purged from these formulations by the use of EOF, the gel matrices have limited structural stability in useful chromatographic solvents such as acetonitrile. Moreover, polyacrylamide gels are highly swelled gels of low polymer content that rely on the solvent for their structure. Thus, these gels suffer from the draw back that they cannot be dehydrated without losing their structure.
A method for separating and concentrating charged species from uncharged or neutral species regardless of size differential has been disclosed by Singh et al. in U.S. patent application Ser. No. 09/256,586 entitled Electrokinetic Concentration of Charged Molecules, incorporated herein in its entirety. The method uses reversible electric field induced retention of charged species, that can include molecules and molecular aggregates such as dimers, polymers, multimers, colloids, micelles, and liposomes, in volumes and on surfaces of porous materials. The retained charged species are subsequently quantitatively removed from the porous material by a pressure driven flow that passes through the retention volume and is independent of direction thus, a multi-directional flow field is not required. Uncharged species pass through the system unimpeded thus effecting a complete separation of charged and uncharged species and making possible concentration factors greater than 1000-fold.
Singh et al. have found that the phenomenon of retention or trapping of charged molecules under the influence of an electric field occurs neither in an open capillary or channel nor in a capillary or channel packed with a stationary phase consisting of nonporous silica or polymer particles having a diameter of 1 μm or larger. Consequently, the porous stationary phase in Singh et al. has certain required properties. First, it must be capable of supporting electroosmotic flow. Second, the porous particulate material must have certain physical characteristics in order to effectively trap and retain charged particles. Silica particles having a diameter of about 1.5 to 20 μm and containing pores having a diameter of about 50 to 500 Å are preferred as a stationary phase material and silica particles having a diameter of about 5 μm and containing pores having a diameter of about 300 Å are particularly preferred.
While a porous particulate stationary phase has been shown to effectively trap and retain charged particles, these stationary phases are very difficult to fabricate, particularly in microchannels. In particular, the need to retain the particulate materials within the chromatography column requires fabrication of porous frits of controlled pore size over a significant length and high mechanical stability. This requirement presents a significant challenge to the use of a porous particulate stationary phase for separation of charged from uncharged species.